Space and time in the nucleus: developmental control of replication timing and chromosome architecture.

All eukaryotic cells replicate segments of their genomes in a defined temporal sequence. In multicellular organisms, at least half of the genome is subject to changes in this temporal sequence during development. We now know that this temporal sequence and its developmentally regulated changes are conserved across distantly related species, suggesting that it either represents or reflects something biologically important. However, both the mechanism and the significance of this program remain unknown. We recently demonstrated a remarkably strong genome-wide correlation between replication timing and chromatin interaction maps, stronger than any other chromosomal property analyzed to date, indicating that sequences localized close to one another replicate at similar times. This provides molecular confirmation of long-standing cytogenetic evidence for spatial compartmentalization of early- and late-replicating DNA and supports our earlier model that replication timing is reestablished in each G(1) phase, coincident with the anchorage of chromosomal segments at specific locations within the nucleus (timing decision point [TDP]). Here, we review the evidence linking the replication program to the three-dimensional architecture of chromatin in the nucleus and discuss what such a link might mean for the mechanism and significance of a developmentally regulated replication program.

Mapping sites where replication initiates in mammalian cells using DNA fibers.

DNA replication in mammalian chromosomes takes place as a unit of replicon clusters. Here we show a powerful method to detect replication origins and fork movement on DNA fibers from mammalian cells. Cells were loaded with nucleotide analogs, DNA fibers were prepared, and replicated DNA was detected. Using this approach, we could detect origins as close as 10 kb apart and found that the average size of replicon is smaller ( approximately 46 kb) than previously estimated. In addition, the procedure visualizes the complex structure of replicon clusters, e.g. sequential activation of origins in a cluster and flexible initiation sites in different cell cycles. Combined with fluorescence in situ hybridization, replication origins can be mapped in genomic loci including repetitive DNA and a single-copy gene.